Microfluidic devices and methods promise breakthrough applications in biotechnology such as directed evolution, tissue printing, and bead-based PCR in emulsions, while facilitating many quantitative studies of biology at a single-cell level. For example, in some microfluidic methods, individual cells can be made to reside within separate picoliter-volume liquid drop, chemically isolated from other droplets. This can permit cell-secreted molecules to rapidly achieve detectable concentrations in a confined fluid droplet surrounding the encapsulated cell. Similarly, uptake of trace chemicals specific to individual cells can be probed due to their depletion within the confined extracellular fluid. Moreover, highly monodisperse droplets of water in an inert and immiscible carrier fluid can be formed at rates of several kHz using microfluidic techniques. These droplets can be combined in pairs, split in two, and selected based on the contents of individual droplets. However, variability in the number of cells or other particles per drop of fluid due to stochastic cell loading is a major barrier to an effective use of these techniques.
Existing processes for loading individual cells into droplets are typically random processes with the distribution of the number of cells in each droplet being dictated by Poisson statistics. Accordingly, the probability of a drop containing k cells is λk exp(−λ)/(k!), where k is the average number of cells per drop. The ratio of droplets containing one cell to those containing two is 2/λ. This means that to minimize the number of droplets that contain more than a single cell requires very low average loading densities. As a result, most droplets actually contain no cells whatsoever. This constraint significantly reduces the number of usable droplets. For example, only 15.6% of all droplets will contain one cell if no more than one in ten of the occupied droplets can be allowed to hold two or more cells. There is a need for microfluidic devices and methods for forming a higher proportion of liquid droplets containing a single cell.